Priapism associated with sickle cell disease affects several thousand men in the US and millions worldwide. At present there are no satisfactory medical treatments to prevent the onset of priapism in these patients, in part because the mechanisms involved in the development of priapism are complex and not well understood. Our group has established that a family of pentapeptides called opiorphins plays a role in the development of priapism. In previously published studies we have demonstrated that over-expression of the opiorphin gene in the rat corpora by gene transfer results in a priapic-like state, and in an animal model of sickle cell disease (the Berkley sickle cell (BERK) mouse) there is elevated expression of the mouse opiorphin genes in corporal tissue prior to the development of priapism (which occurs regularly in these animals). Our recent work has discovered that opiorphins are up-regulated in hypoxic conditions, and that opiorphins can regulate hif1a and a2br expression, two genes that have previously been associated with smooth muscle relaxant pathways and priapism. This has led us to propose the following hypothesis: Sickle cell disease causes hypoxia/ischemia in corporal tissue which results in increased opiorphin expression. Opiorphin then acts as a master regulator of compensatory smooth muscle relaxant pathways. It is the excessive activation of these relaxant pathways that could then result in priapism. Understanding the association between opiorphin activation in corporal tissue as a result of sickle cell disease may lead to the development of prognostic markers and novel pharmacological strategies to prevent or treat priapism associated with sickle cell disease. In order to test this hypothesis we propose the following three specific aims: In the first specific am we will use an in vitro model to determine the effect of hypoxia on expression of opiorphins and other genes involved in the development of priapism or regulation of smooth muscle relaxant pathways in human and mouse corporal smooth muscle cells. We will determine which of these pathways are directly regulated by opiorphins. We will also use a mouse model of corporal hypoxia to determine if similar response mechanisms operate in vivo. In the second specific aim we will use a BERK mouse model of sickle cell disease to determine if up-regulation of opiorphins expression is an early event prior to the development of priapism, and if similar pathways are activated as in the hypoxic models. We will distinguish which of these pathways are causative rather than responsive to the physiological condition of priapism by using chemical induction of priapism in the mouse. In patients we will compare circulating levels of opiorphin in sickle cell and normal men, and determine if blood levels of opiorphin correlate with the risk of developing priapism. In the third specific aim we will use mouse models to directly demonstrate that opiorphins play a role in erectile function. We will use intracorporal gene transfer to over- express opiorphin genes in the mouse, and determine if this will result in a priapic-like condition. We will generate conditional knockouts of the mouse opiorphin genes, and determine the effect on erectile function when the opiorphin gene is knocked-out. We will also cross the opiorphin knockout mice with the BERK sickle cell mice to determine if opiorphin expression effects the development of priapism in sickle cell mice. Our research will potentially identify novel pharmacological targets for the diagnosis, prognosis, prevention and treatment of priapism associated with sickle cell disease.